Antimicrobic compositions and use thereof



3,223,547 ANTlB/HCROBHC C(DMPOSITlONS AND USE THEREQF Jakob Bindler,Riehen, near Basel, and Ernst Model,

Basel, Switzerland, assignors to Geigy Chemical Corporation, Ardsley,N.Y., a corporation of Delaware No Drawing, Original application .lune6, 1962, Ser. No. 280,355. Divided and this application Apr. 10, 1964,Ser. No. 367,269 Claims priority, application Switzerland, June 7, i961,6,605/61 9 Claims. (Cl. 106-316) This application is a division ofSerial No. 200,355, filed June 6, 1962.

The present invention concerns antimicrobic compositions and their usefor the cpn l a ttjpg gf bactgri a and, possibly, als o fpngi and forthe protection of organic materials and objects from attack primarily bybacteria and, possibly, also fungi, as well as, as industrial product,the organic materials protected by the aid of these antimicrobic agentsprimarily from attack by bacteria (and possibly also from attack byfungi or from rot);

By antimicrobic agents in the present application, protective agentsagainst bacteria are meantwhich contain active ingredients havingbactericidal or bacteriostatic properties which active ingredients inaddition also have more or less marked fungicidal or fungistaticproperties.

The antimicrobic compositions according to the invention contain, asactive ingredients, aromatic acid amide derivatives of the generalformula 3 C 011201 O RX-NH- J L Y2 I1 Yr (1) wherein n is the number 0or 1,

Y is hydrogen or a chlorine atom,

Y is hydrogen or, if Y is hydrogen, it can also be a chlorine atom,

Z is hydrogen or the chloroa'cetyl group COCH Cl,

R is an alkyl radical, if desired monochloroor polychloroalkyl having 1to 3 carbon atoms or, if n is 0, it can also be a phenyl radical, ifdesired monochloroor polychloro-phenyl, the amino group, an alkylaminoradical having at most 5 carbon atoms, a dialkylamino radical, having intotal at most 5 carbon atoms, or a phenylamino radical, the latter ifdesired monosubstituted by a monochloroacetyl group, and

X is the -CO group and, if R is an alkyl radical, X can also be the SOgroup.

These active ingredients have excellent antimicrobic, in particularbactericidal or bacteriostatic properties, and in addition can also havefungicidal or fungistatic activity. They are only slightly toxic forwarm blooded animals, they have no irritant action on the skin and arethus excellently suitable as active substances for the combatting ofbacteria and fungi as well as for the protection of organic materials,including the human skin, from attack by bacteria and fungi and for theprotection of organic materials from injury by rot.

Preferred active substances are those in which X is the carbonyl groupC0-.

The following preferred chloroacetylated acid amide derivativesaccording to the above definition are employed as antimicrobic activesubstances to be used according to the invention:

(a) Chloroacetylated, aliphatic or aromatic carboxylic acid or sulphonicacid anilides, particularly acetanilides, of the restricted generalformula Active substances of the general Formula II are, for example:

4-chloroacetyl-acetanilide, 4-chloroacetyl-Z-chloracetanilide,4-chloroacetyl-3-chloroacetanilide, 4-chloroacetyl-u-chloracetanilide,4-chloroacety1-4x, x-dichloracetanilide,4-chloroacetyl-or,a,a-trichloracetanilide,4-chloroacetyl-n-butyrylanilide,4-chloroacetyl-(p-chlorobenzoyl)-anilide,4-chloroacetyl-methylsulphanilide,

(b) Chloroacetylated diphenyl ether amides of the restricted generalformula R2-XNH (III) wherein X represents the CO or --SO group,

R represents an alkyl radical, if desired monochloroor polychloroalkylhaving at most 3 carbon atoms, preferably a methyl radical ormonochloromethyl or polychloromethyl and Y Y and Z have the meaningsgiven above.

Examples of active substances of the general Formula III are thefollowing:

4-chloroacetyl-4'-acetamido-diphenyl ether,4-chloroacetyl-4-chloro-2-acetamido-diphenyl ether,4-chloroacetyl-4'-chloroacetamido-diphenyl ether,4-chloroacetyl-2'-chloro-4-acetamido-diphenyl ether,4-chloroacetyl-2'-chloro-4-chloroacetamido-diphenyl ether,4,5-bis-chloroaceyl-2-chloro-2'-acetamido-diphenyl ether,4-chloroacetyl-4-methane sulphonamido-diphenyl ether.

(0) Chloroacetylated urea derivatives of the restricted general formulaY1 (IV) wherein Y, represents hydrogen or chlorine, R representshydrogen, an alkyl radical with up to 5 carbon atoms or a phenyl radicalor a phenyl substituted by at most one ClCH CO, and

R represents hydrogen or a low alkyl radical, whereby alkyl radicals Rand R together should not contain more than 5 carbon atoms.

The following compounds are given as examples of active substances ofthe general Formula IV:

N-(4-chloroacetylphenyl -urea, N-n-butyl-N'- (4-chloroacetylphenyl-urea, N-diethyl-N- (4-chloroacetylphenyl) -urea,N,N-bis-(4-chloroacetylphenyl) -urea,N-phenyl-N-(4-chloroacetylphenyl)-urea,N-n-amyl-N'-(4-chloroacetylphenyl)-urea.

Those of the active substances to be used according to the invention canbe easily produced by known methods.

In general, they are obtained from carboxylic or sulphonic acid anilidesor N-phenyl ureas or diphenyl ether amides, possibly substituted asdefined, by introduction of the chloroacetyl group with chloroacetylchloride in the presence of AlCl according to Friedel-Crafts.

Also such acid anilides or phenyl ureas can be reacted with acetylchloride according to Friedel-Crafts to introduce the acetyl group intothe aromatic nucleus thereof and then the acetyl group can bechlorinated.

Vice versa, correspondingly substituted anilines or diphenyl etheramines can be used as starting materials and the amino group can beconverted into an acid amide group by acylation or a chloroacetylanilinecan be converted in the known manner into a urea derivative, e.g., byreaction with suitably substituted isocyanates or carbamic acidchlorides.

In general, the active substances according to the invention arecolourless to pale yellowish coloured solid bodies which can be purifiedby recrystallization. They are only slightly soluble in water butdissolve in the usual organic solvents. These solubility properties inorganic solvents which are used for so-called dry cleaning, enable theactive substances to be used as organic solutions in the form of spraysor as dry cleaners in the presence of neutral to weakly alkaline wettingand cleansing agents. Also the active substances caghe gdmixed...

wwith p apg treatmentdiqugrs or printing thickeners rnade from starch orcellulose derivativs'or they can be used for the impregnation of wood.Also in objects made from synthetic resins such as, e.g., polyvinylchloride films, etc. as well as in lacquers and paintsmrhich contain,e.g., casein or synthetic resins, an effective protection can beattained with the substances to be used according to the inventionagainst attack by bacteria and, possibly, fungi.

The active substances given above and others of the general Formula I orof the restricted general Formulae II to IV are distinguished by onlyslight toxicity to warm blooded animals and are, in practice,non-irritant to skin and eyes. These active substances can also be usedin synthetic washing and cleansing agents in solid form such as tabletsor bars, which agents contain, for example, non-ionogenic products orfatty acid condensation products as wash active component. But theactive substances can also be used in anion-active or cation-activewashing and cleansing agents such as soda soaps, etc. The activesubstances according to the invention have an excellent action inparticular against bacteria, both gram positive as well as gram negativesuch as, e.g., Escherichia coli. They can also have a fungicidal actionwhich, depending on the active substance, is more or less marked; someof the active substances have a very good action against fungi.

Another advantage is their lack of, or only pale, colour, which propertyenables them to be used for many purposes for which the known stronglycoloured compounds cannot be used.

The antimicrobic active substances can be applied to the organicmaterial to be protected either by simple admixture or by impregnationwith solutions or suspensions of the active substance. Contents of 5 to20 grams (g.) of active substance per litre treatment liquor aregenerally sufiicient to attain effective protection of the sprayed orimpregnated material against attack by bacteria and fungi, rot, orformation of mildew spots. Organic material containing 0.5 to 2% ofactive substance, calculated on the weight of the material, is generallyeffectively and lastingly protected against attack by bacteria and,

possibly, also fungi. 5&nig material to be protected; are chieflycellulose materias sucli as'b'ellfilhe'fwood,

paper, materials made from synthetic resin as well as lacquers andpaints. However, also other organic substances which tend to mould orrot can be protected such as leather, vegetable or animal mucilages andjells, permanent sizings made from polyvinyl, etc.

The good activity of the active substances to be used according to theinvention against bacteria can be seen from the following laboratorytest:

By producing solutions of varying concentration of the active substanceto be tested in ethylene glycol monomethyl ether (methyl Cellosolve) andaddition of 1.25% of these methyl Cellosolve solutions to 20 ml. agar,nutrient media are produced which contain the following 8 finalconcentrations of active substance in p.p.m. (=parts of active substanceper 10 parts substratum): 300; 30; 10, 3; 1; 0.3 and 0.1.

After solidifying, the agar nutrient media are inoculated with 24 hourold cultures of the following strains of bacteria:

Staphylococcus aurens SG 511 Escherichia coli 96 Bacillus mesentericusSarcina lutea The dishes containing the agar media injected withbacteria are kept for 48 hours at 37 and the growth of the organisms onthe agar is adjudged visually. The following table gives the results ofthe test, in which the lowest concentration of active substance inp.p.m. (:parts of active substance per 1 million parts of carriersubstance) is given at which complete inhibition of growth can beobserved.

The following active substances were used for this test:

TABLE Substance Staph.aureus Eschcoli Bac. mes. I Sara. Zut.

From the above table the uniformly wide action spectrum and theexcellent action of the substances used according to the invention,which also covers gram-negative bacteria such as, e.g. Escherichia coli,can be seen.

The active substances of the general Formula I according to theinvention can also be used in combination with other fungicidal and/orbactericidal substances, e.g., to-

closed and the culture is incubated for days at 28 C. The development ofa fungi growth is then judged:

Results:

Concentration of active substance Active substance4-chloroacetyl-acetanilide No growth Slight growth Moderate growth.Control without active substance Strong growth Strong growth Stronggrowth.

gether with halogenated salicylic acid alkylamides and anilides,halogenated diphenyl ureas, halogenated benzoxazolones,polychlorohydroxydiphenylmethanes, halogenated dioxy-diphenyl sulphidesetc.

On using the active substances according to the invention simultaneouslywith other bactericidal or fungicidal compounds, often a better actioncan be determined than would have been expected from the sum of the twoeffects (synergism).

In the bacteriostatic test, mixtures of active substances according tothe invention such .as, e.g. 4-chloroacetylacetanilide and4-chloroacetyl-4'-chloroac-etamidodiphenyl ether with knownbacteriostatica such as 3-trifluoromethyl-4,4'-dichlorodiphenyl urea,3,4,4'-trichloro2'-hydroxydiphenyl urea etc. clearly show a synergisticincrease in action. 4 chloroacetyl 4' chloroacetamidodiphenyl ethermixed with 2-hydroxy-3,5-dichlorobenzoic acid-3,4- dichloranilide orwith hexachlorophene (2,2'-dihydroxy- 3,5,6,3,5',6-hexachloride phenylmethane) also has a clear increase of action.

In the following application examples, parts are given as parts byweight where not otherwise expressly stated; percentages are to beunderstood as percentages by weight and the temperatures are in degreesCentigrade.

Example 1 .Paint 130 parts of an emulsion paint having a 75% content ofsolid substances and consisting of 36 parts of chalk 9 parts of titaniumdioxide 22.5 parts of Mowilith D 025 (Hoechst) 0.5 parts of Belloid TD10 parts of pigment paste of about 60% solid component ad 100 parts ofwater are mixed with 8 parts of a 25% stock solution of 4-chloroacetyl-acetanilide, ethylene glycol monomethyl ether and dimethylformamide 1:1 serving as solvents. This produces a paint which contains2% of active substance calculated on the solid content. In the same way,using 1.6 -or 0.32 part of active substance, paints are produced whichcontain 0.4 and 0.08% respective of 4-chloroacetylacet-anilidecalculated on the solid content.

Pieces of cardboard made from wood pulp of about 1 mm. thickness and 6 x6 cm. square are so painted on both sides that, per sq. m. of surface200 g. of each of the 3 previously prepared mixtures are evenly applied(=1.44 g./72 cm. The cardboard pieces are dried for some days and thensubjected to the biological test in the following way:

A pumice stone plate of 6 x 6 x 0.5 cm. is laid in a petri dish of 10cm. diameter and covered with a 6 x 6 cm. glass plate. The pumice stoneplate is saturated with distilled water. The piece of cardboard to betested is laid on the glass plate and inoculated with 1 ccm. of asuspension of spores of the following composition:

Inoculum: 10 ccm. of Czapek solution are added to 14- day-old slant agarculture of the fungus Trichoderma viride and the surface of the cultureis carefully scraped and suspended; it is then filtered throughfibreglass and filtrate is used as inoculum. The petri dishes are then15 Example 2.-Plasticizcr for synthetic resins The active substances,4-chloroacetyl-acetanilide is dissolved in ethylene glycol monomethylether (methyl Cellosolve) in suitable concentrations.

4 parts of this solution are mixed with 5 parts of dioctyl sebacate. Theinsensitivity of the plasticiser so obtained to fungal and bacterialattack is tested as follows: This plasticiser mixture is added to 91parts of nutrient agar and the agar mixture is poured into plates. Aftersolidifying, inoculation is made with a l4-day-old culture ofAspergillus oryzae or with a 24-hour-old culture of Escherichia coli 96.The inoculum is obtained as follows:

10 ccm. of physiological sodium chloride solution are added to each of14-day-old slant agar cultures of Aspergillus oryzme or of the24-hour-old slant agar cultures of Escherichia coli 96. The surface ofthe cultures is carefully scraped and suspended. The suspension is thenfiltered through fibreglass and the filtrate is used as inoculum.

The nutrient agar for Aspergillus oryzae is Sabouraud- Maltose:mineralsalt agar 1:20. If Escherichia coli 96 is inoculated, the nutrient agaris nutrient agarzmineral salt agar=1 :20.

The methyl Cellosolve solutions contain so much active substance thatthe concentrations, calculated on the plasticiser, are 2.0%, 0.4% and0.08%. The plates inoculated with Aspergillus oryzae are left for 10days at 28 C., whilst those inoculated with Escherichia coli 96 are keptfor 48 hours at 37 C. The development of the organisms on the agarsurface is then judged.

TABLE Concentrations Aspergillus oryzae Escherichia coli 96 No growth. 7Do. 0.08%. Growth. Control without Do.

active substance.

Example 3 Washing liquor for various textile fibres. The activesubstance, N n butyl N (4 chloroacetyl phenyl) urea is added inconcentrations of 0 mg. and 100 mg. per litre to a soap solutioncontaining 1.5 grams (g.) of anionactive soda-soap per litre. Cottonfabric, wool fabric or nylon staple fabric which has been inoculatedwith 24-hour-old Staphylococcus aureus SG 511 bacteria, cultured in abroth medium, and which fabric has then been incubated several hours, isentered into the washing liquor in the liquor ratio of 1:20 for 20minutes at 40 C. On finishing the washing procedure, 1 ml. of washingliquor is mixed with 15 ml. of 45 C. warm agar medium and the agar ispoured into plates immediately. A sample of the textile material is alsotaken, spread on a sterile agar plate and incubated for 48 hours.

Thereupon the above fabric is rinsed twice in softened, cold water in aliquor ratio of 1:20 for 3 minutes, then wrung and dried.

Circles 20 mm. in diameter are cut from the prepared fabric and spreadon agar plates which have been prewherein viously inoculated withStaphylococcus aureus SG 511. The plates are then incubated for 48hours.

After incubation, it is observed that the washing liquor containing theactive substance is sterile, whereas that which do not contain theactive substance contains a large number of bacteria per ml.Furthermore, those agar plates whereon the textile material has beenplaced immediately after washing with the liquor containing N- n butyl N(4 chloroacetyl phenyl) urea show no bacteria colonies, whereas thosetextile fabrics which have not received the active substance are loadedwith bacteria. Fnally, those agar plates which have previously beeninoculated with Staphylococcus aureus SG 511 and then covered withfabric discs made of fabric which has been washed in the presence of theactive substance, show 15 been washed in the presence ofN-n-butyl-N'-(4-chloro- 20 acetyl-phenyl)urea, and which show no zoneswherein bacterial growth is inhibited, furthermore, such fabric discsare covered with bacterial colonies.

wherein R is a member selected from the group conslsting of alkylcontaining at most three carbons, monochloroalkyl containing at mostthree carbons and polychloroalkyl containing at most three carbons,phenyl and monochlorophenyl,

Y is a member selected from the group consisting of hydrogen andchlorine and X is a member selected from the group consisting of -CO andSO with the limitation that if X is -SO R must be a member selected fromthe group consisting of said alkyl, said monochloroalkyl and saidpolychloroalkyl, said component (b) being present in an amount etfectivefor protecting said plasticizer against microbial attack.

3. An emulsion paint consisting essentially of (a) a pigment componentfor imparting color to said paint,

(b) an aqueous medium in which said pigment component is emulsified,

(c) a synthetic resin base for said pigment, which resin base issensitive to microbial attack, and

Cone. of N-n-butyl- Bacteria count Bacteria on textileN-(4-ehloroacetyl- Fabric per m1. sample after the phenyD-urea in thewashing liquor washing process washing liquor Width in mm.

of the inhibi- Bacterial colonies tion around on the disc the 20 mm.disc app. 70, 000 Large number- 10 0 Large number 0 None Large number.None. Large number. None. Large number. None.

What is claimed is: 1. An emulsion paint consisting essentially of (a) apigment component for imparting color to said paint,

(b) an aqueous medium in which said pigment component is emulsified,

(c) a synthetic resin base for said pigment, which resin base issensitive to microbial attack, and

(d) an antimicrobially active compound of the formula macarnQ-ooomm R isa member selected from the group consisting of alkyl containing at mostthree carbons,

monochloroalkyl containing at most three car bons, and polychloroalkylcontaining at most three carbons, phenyl and monochlorophenyl,

Y is a member selected from the group consisting of hydrogen andchlorine,

X is a member selected from the group consisting of CO and SO with thelimitation that if X is SO R must be a member selected from the groupconsisting of said alkyl, said monochloroalkyl and said polychloroalkyl,said component (d) being present in said paint in an and (b) anantimicrobially active compound of the formula (d) an antimicrobiallyactive compound of the formula wherein Y is a member selected from thegroup consisting of hydrogen and chlorine,

R is a member selected from the group consisting of hydrogen, alkylcontaining at most five carbons, phenyl and phenyl substituted by oneClCH CO--, and

R is a member selected from the group consisting of hydrogen and alkylcontaining at most five carbon atoms with the limitation, as to R and Rthat the total number of carbons in the alkyl radicals, when both R andR are an alkyl, is at most five, said component (d) being present insaid paint in an amount effective for protecting said paint againstmicrobial attack.

4. A plasticizer composition consisting essentially of (a) an organicplasticizer sensitive to microbial attack, and (b) an antimicrobiallyactive compound of the formula wherein Y is a member selected from thegroup consisting of hydrogen and chlorine,

R is a member selected from the group consisting of hydrogen, alkylcontaining at most five carbons, phenyl and phenyl substituted by oneClCH CO, and

R is a member selected from the group consisting of hydrogen and alkylcontaining at most five carbon atoms with the limitation, as to R and Rthat the total number of carbons in the alkyl radicals, when both R andR are an alkyl, is at most five, said component (b) being present in anamount effective for protectting said plasticizer against microbialattack.

5. An emulsion paint as defined in claim 1, wherein said synthetic resinbase consists of polyvinyl acetate.

6. An emulsion paint as defined in claim 5, wherein said antimicrobiallyactive compound is 4-chloroacetyl acetanilide.

7. An emulsion paint as defined in claim 3, wherein said antimicrobiallyactive compound is N-n-butyl-N'-(4- chloroacetyl-phenyl)-urea.

8. A plasticizer composition as defined in claim 2, wherein saidplasticizer is dioctyl sebaceate.

9. A plasticizer composition as defined in claim 8, wherein said'antimicrobially active compound is 4-chloroacetyl acetanilide.

References Cited by the Examiner Elliott et al., J. Chem. Soc., 1949,pages 552-555.

MORRIS LIEBMAN, Primary Examiner.

2. A PLASTICIZER COMPOSITION CONSISTING ESSENTIALLY OF (A) AN ORGANICPLASTICIZER SENSITIVE TO MICROBIAL ATTACK, AND (B) AN ANTIMICROBIALLYACTIVE COMPOUND OF THE FORMULA
 3. AN EMULSION PAING CONSISTINGESSENTIALLY OF (A) A PIGMENT COMPONENT FOR IMPARTING COLOR TO SAIDPAINT, (B) AN AQUEOUS MEDIUM IN WHICH SAID PIGMENT COMPONENT ISEMULSIFIED, (C) A SYNTHETIC RESIN BASE FOR SAID PIGMENT, WHICH RESINBASE IS SENSITIVE TO MICROBIAL ATTACK, AND (D) AN ANTIMICROBIALLY ACTIVECOMPOUND OF THE FORMULA